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Mechanisms of induction and propagation of cortical spreading depression in mouse brain slices
Full text linkMigraine is a common and highly disabling neurological disorder that affect more than 10% of the general population; in 30% of the migraneours migraine headache is preceded by transient neurological symptoms referred as “migraine aura”. Familial Hemiplegic Migraine type 1 (FHM1) is a rare and very severe autosomal dominant subtype of migraine with aura (MA), that is considered a good model for studying migraine: FHM1 typical attacks resemble general MA attacks, but aura symptoms include also motor weakness or paralysis, often but not always unilateral. FHM1 is caused by mutations in the CACNA1A gene, with encodes the α1, pore-forming subunit of neuronal CaV2.1 voltage-gated Ca2+ channels, also referred as P/Q-type Ca2+ channels.
FHM1 mutations produce gain-of-function of human recombinant CaV2.1 channels, mainly due to a shift of channel activation to more negative voltages and an increase of the open probability and single channel influx over a wide voltage range (Hans et al., 1999; Tottene et al., 2002, 2005; Pietrobon).
Facilitation of experimentally induced cortical spreading depression (CSD) in knock-in (KI) mice carrying the human FHM1 mutation R192Q has been observed in vivo (van den Maagdenberg et al., 2004) and in vitro (Tottene et al., 2009). CSD, a wave of strong neuronal and glial depolarization that slowly progresses across the cerebral cortex, followed by long-lasting neural suppression, is the phenomenon underlying migraine aura and a likely primary cause of migraine headache.
Tottene et al. (2009) recently demonstrated enhanced excitatory neurotransmission due to increased action potential-evoked Ca2+ influx through CaV2.1 channels and increased probability of glutamate release at cortical pyramidal cell synapses of R192Q KI mice. Using an in vitro model of CSD Tottene et al. also demonstrated a causative link between enhanced glutamate release and facilitation of CSD induced by brief pulses of high KCl in cortical slices of KI mice. These findings support a key role of CaV2.1 channels in CSD initiation and propagation.
In the first part of my Ph. D project I further investigated the mechanisms of CSD induction and propagation and in particular the role of NMDA receptors and voltage-gated calcium channels. Results obtained were published in Tottene el al., 2011 (pdf attached in chapter 3). We adopted the in vitro model of CSD described in Tottene et al., 2009 and measured CSD threshold and velocity in acute cortical slices from WT mice before and after application of specific blockers. CSD was induced by pressure pulses of high KCl of increasing duration; the duration of the first pulse eliciting a CSD was taken as CSD threshold and the rate of horizontal spread of the change in intrinsic optical signal produced by CSD as CSD velocity. We investigated the role of NMDA using the general NMDA receptors inhibitor D-AP5 (50 μM). In the presence of D-AP5 stimuli up to 30-time the duration of the control threshold failed to induce a CSD. We investigated also the role of P/Q, N, R, and L-type voltage-gated calcium channels using the respective specific blockers: ω-agatoxin IVA (300 nM), ω-conotoxin GVIA (1 μM), SNX-482 (250 nM), and nimodipine (50 μM). Similarly to what observed after application of D-AP5, in the presence of ω-AgaIVA stimuli up to 30-time the duration of the control threshold failed to induce a CSD. Application of either ω-CgTxGVIA and SNX-482 only slightly increased CSD threshold (of about 10%) and slightly decreased CSD velocity (of about 15%) suggesting that N- and R-type voltage-gated Ca2+ channels may have a modulatory role in CSD induction and propagation. Application of nimodipine did not affect CSD induction nor propagation. The findings that application of D-AP5 or ω-AgaIVA inhibits CSD induction while ω-CgTxGVIA, SNX-482 and nimodipine have only a poor or null effect are consistent with and support a model of CSD initiation and propagation in which activation of presynaptic (and possibly postsynaptic) CaV2.1 channels and NMDA receptors plays a pivotal role (Tottene et al., 2011).
The role of NMDA receptors was further investigated in FHM1 R192Q mice, which show an increased glutamate release from pyramidal neurons. In contrast to what happens in WT mice, application of D-AP5 in brain slices from KI mice did not prevent CSD induction, but significantly increased CSD threshold (90 ± 9%) and significantly decreased CSD velocity (47 ± 1%) relative to control. The different effect of D-AP5 in WT and KI mice suggests that other elements (beside NMDA receptors) could be involved in CSD initiation mechanisms.
Current clamp recordings close to the KCl injection pipette (at fixed distance of 100 and 200 μm) allowed to observe different phases in voltage changes after the application of both sub-threshold and threshold KCl stimuli: it was possible, in fact, to distinguish three distinct peaks. While the first two peaks were present in both sub-threshold depolarizations and CSD, the third peak was specifically linked to CSD. Patch-clamp technique allowed to accurately describe different CSD phases but showed the obvious limitation to be restricted to only one cell. Ca2+ imaging of slices loaded with the Ca2+ sensitive indicator OGB1-AM allowed to observe several neurons at different distances from KCl-injecting pipette at the same time for each pulse. Simultaneous patch-clamp recordings and Ca2+ imaging showed a general correlation between voltage changes recorded in current-clamp and fluorescence peaks detected in Ca2+ traces. The physiological meaning of these different phases and their specific role in CSD induction and propagation are not clear and are currently object of study in our laboratory.
Ca2+ imaging allowed to detect also intracellular calcium increases from astrocytes in response to KCl pulses. Astrocytes can support and amplify glutamatergic synaptic transmission by releasing glutamate that specifically act on NMDA receptors; astrocytic release of glutamate and other substances is induced by [Ca2+]int increases that are, in turn, stimulated by neuronal release of glutamate and other neurotransmitters. To test whether the neuron-astrocyte crosstalk is involved in CSD induction and propagation, threshold for CSD induction and velocity of CSD propagation were measured before and after inhibiting astrocytic [Ca2+]int increases by the application of cyclopiazonic acid (CPA, 50 μM), that emptied astrocytic Ca2+ internal stores without affecting neuronal response to KCl. CPA application did not affect CSD threshold or velocity in both WT and KI mice. These results show that neuron-astrocyte crosstalk seem not to be relevant in CSD induction and propagation.
The combination of Ca2+ imaging and patch clamp recording permitted to observe, in the absence of external stimuli, spontaneous activity that occurs in the neocortical neural networks as slow oscillations between periods of synaptically-driven depolarizations with firing (up-states) followed by decreased synaptic inputs and ceasing of firing (down-states). At the same time spontaneous large Ca2+ oscillations were observed in astrocytes. To check whether neuron-astrocyte crosstalk could be involved in the generation or in the modulation of spontaneous neuronal network activity, astrocytic spontaneous oscillations were inhibited by application of CPA. In presence of CPA up-states were still present in both WT and KI mice; however CPA application led to a significant reduction of up-states frequency in WT (41 ± 3%, p = 0.0001), but not in KI mice (11 ± 6%, p = 0.12). These preliminary results are in agreement with a possible role of neuron-astrocyte crosstalk in spontaneous neuronal network activity.L’emicrania è un disturbo neurologico comune ed altamente invalidante che colpisce più del 10% della popolazione in generale; nel 30% dei pazienti affetti da emicrania, la fase del dolore è preceduta da sintomi neurologici transienti definiti nel complesso “aura emicranica”. L’emicrania emiplegica familiare di tipo 1 (FHM1) è una forma autosomica dominante di emicrania con aura rara ma molto grave; l’FHM1 è considerata un buon modello per lo studio dell’emicrania: gli attacchi tipici di FHM1, infatti, ricapitolano molto quelli della normale emicrania con aura, tuttavia durante l’aura si presentano sintomi come debolezza motoria o paralisi, spesso ma non sempre unilaterale. L’FHM1 è dovuta a mutazioni nel gene CACNA1A che codifica la subunità α1 formante il poro dei canali Ca2+ neuronali voltaggio dipendenti CaV2.1, chiamati anche canali per il calcio di tipo P/Q.
Le mutazioni FHM1 producono un guadagno di funzione dei canali ricombinanti umani CaV2.1; tale guadagno di funzione è principalmente dovuto allo spostamento della curva di attivazione del canale verso valori di potenziale più negativi e all’aumento della probabilità di apertura e dell’influsso di Ca2+ a livello di singolo canale in un ampio intervallo di potenziali vicini alla soglia di attivazione del canale (Hans et al., 1999; Tottene et al., 2002, 2005).
Studi in vivo (van den Maagdenberg et al., 2004) ed in vitro (Tottene et al., 2009) hanno dimostrato come topi knock-in (KI) per la mutazione umana FHM1 R192Q presentino una facilitazione nell’induzione sperimentale della cortical spreading depression, il fenomeno neurologico alla base dell’aura emicranica e possibile evento innescante il tipico mal di testa caratterizzante l’emicrania. Dal punto di vista elettrico la CSD è un’onda di forte depolarizzazione neuronale e gliale, che si propaga lentamente attraverso la corteccia cerebrale ed è seguita da una lunga fase di soppressione neurale.
Recentemente Tottene e colleghi (2009) hanno dimostrato un aumento nella trasmissione sinaptica eccitatatoria corticale dei topi FHM1 R192Q; tale aumento è dovuto all’aumentato influsso di Ca2+ evocato da potenziali d’azione attraverso i canali CaV2.1 ed al conseguente aumento nella probabilità di rilascio del neurotrasmettitore glutammato alle sinapsi piramidali di questi topi. Attraverso l’impiego di un modello in vitro, Tottene e colleghi hanno, inoltre, dimostrato il rapporto di causalità tra l’aumentato rilascio di glutammato dai neuroni piramidali e la facilitazione nell’induzione della CSD tramite brevi applicazioni pressorie di KCl a concentrazioni elevate in fettine di cervello da questi topi. Tali evidenze supportano l’ipotesi di un ruolo chiave dei canali CaV2.1 nei meccanismi di induzione e propagazione della CSD.
La prima parte del mio progetto di dottorato ha avuto come scopo fondamentale l’approfondimento delle conoscenze riguardo i meccanismi che sottendono induzione e la propagazione della CSD; in particolare il mio interesse si è principalmente soffermato sul ruolo dei canali del calcio voltaggio-dipendenti e dei recettori NMDA. I risultati ottenuti sono stati pubblicati (Tottene et al., 2001, pdf allegato nel capitolo 3). Per conseguire gli scopi prefissi abbiamo impiegato il modello in vitro già in precedenza adottato da Tottene e colleghi (2009) ed abbiamo misurato la soglia di induzione della CSD e la velocità di propagazione della stessa in fettine corticali di topo wild-type (WT) prima e dopo l’applicazione di vari inibitori selettivi dei canali d’interesse. La durata del primo impulso in grado di indurre una CSD veniva considerata la soglia di induzione della stessa, mentre la velocità di propagazione della CSD coincideva con la velocità di propagazione dei cambiamenti nel segnale ottico intrinseco (IOS) associati ad essa. Il ruolo dei recettori NMDA è stato indagato mediante l’impiego di un inibitore selettivo in concentrazione saturante: D-AP5, 50 μM. In presenza di D-AP5 stimoli fino a 30 volte la soglia controllo non erano in grado di indurre una CSD. Abbiamo inoltre indagato il ruolo dei canali per il Ca2+ voltaggio-dipendenti di tipo P/Q, N, R ed L, usando i rispettivi inibitori specifici: ω-agatossina IVA (300 nM), ω-conotossina GVIA (1 μM), SNX-482 (250 nM) e nimodipina (50 μM). Similmente a quanto osservato in presenza di D-AP5, in presenza di ω-AgaIVA, stimoli fino a 30 volte la soglia controllo non erano in grado di indurre una CSD. Diversamente, l’applicazione di ω-CgTxGVIA o di SNX-482 provocava un lieve innalzamento della soglia (di circa il 10%) e una leggera diminuzione della velocità (di circa il 15%), suggerendo un ruolo modulatore dei canali per il calcio voltaggio-dipendenti di tipo N ed R nell’induzione e nella propagazione della CSD. Al contrario l’applicazione di nimodipina non aveva alcun effetto sulla soglia o sulla velocità della CSD. La dimostrazione che D-AP5 e ω-AgaIVA sono in grado di inibire totalmente l'induzione e la propagazione della CSD, mentre CgTxGVIA, SNX-482 e nimodipina presentano solo un lieve effetto, o nessun effetto, sono consistenti con ed avvalorano un modello di induzione e propagazione della CSD nel quale l’attivazione dei canali CaV2.1 presinaptici (e forse anche di quelli postsinaptici) e dei recettori NMDA ha un ruolo essenziale (Tottene et al., 2011).
Il ruolo dei recettori NMDA è stato ulteriormente indagato, sfruttando i topi KI FKM1 R192Q, che presentano un incrementato rilascio di glutammato da parte dei neuroni piramidali. Contrariamente a quanto riscontrato nei WT, in questi topi l’applicazione di D-PA5 non era in grado di bloccare l’induzione della CSD, ma aumentava invece significativamente la soglia (90 ± 9%) e diminuiva significativamente la velocità (47 ± 1%) rispetto al controllo. Il diverso effetto del D-AP5 riscontrato nei topi WT ed in quelli KI potrebbe suggerire che altri elementi (oltre ai recettori NMDA) potrebbero essere coinvolti nell’induzione e nella propagazione della CSD.
Registrazioni in current-clamp in prossimità del sito di eiezione del KCl, alle distanze fisse di 100 e 200 μm, hanno permesso di osservare e distinguere diverse fasi nei cambiamenti di voltaggio indotti dall’applicazione di stimoli di KCl sottosoglia e a soglia; è stato infatti possibile osservare la presenza di tre diversi picchi. Mentre i primi due picchi erano condivisi dalle depolarizzazioni sottosoglia e dalla CSD, la comparsa del terzo picco era specificamente legata all’induzione della CSD. La tecnica del patch-clamp ha permesso di descrivere nel dettaglio le diverse fasi di depolarizzazione, mostrando tuttavia l’ovvia restrizione di limitare l’osservazione ad una sola cellula per volta. Il Ca2+ imaging di fettine caricate con l’indicatore Ca2+ sensibile OGB1-AM ha invece permesso di monitorare allo stesso tempo diversi neuroni a diverse distanze dalla pipetta iniettante KCl dopo lo stesso stimolo. L’esecuzione simultanea di patch clamp e Ca2+ imaging nella stessa fettina ha dimostrato una generale correlazione tra le diverse fasi dei cambiamento di voltaggio registrate in current-clamp ed i picchi nelle tracce di fluorescenza. I possibili significati fisiologici e gli eventuali ruoli nell’induzione e nella propagazione della CSD di queste diverse fasi non sono ancora chiari e sono al momento attuale oggetto di studio nel laboratorio dove ho svolto la mia attività di ricerca.
Gli esperimenti di Ca2+ imaging hanno permesso, oltre a quanto già descritto, di osservare incrementi nella concentrazione intracellulare di Ca2+ degli astrociti in risposta agli stimoli di KCl. Gli astrociti possono sostenere ed amplificare la trasmissione eccitatoria glutamatergica tramite rilascio di glutammato che agisce specificamente sui recettori NMDA; il rilascio di glutammato e di altre sostanze da parte degli astrociti è indotto da innalzamenti nella [Ca2+]int, causati a loro volta dal rilascio di diversi neurotrasmettitori (tra cui il glutammato) da parte dei neuroni. Per verificare se la comunicazione reciproca tra neuroni ed astrociti possa essere coinvolta nell’induzione e nella propagazione della CSD, la soglia e la velocità della CSD sono state misurate prima e dopo l’inibizione degli aumenti di Ca2+ intracellulare tramite applicazione di acido ciclopiazonico (CPA, 50 μM), in grado di svuotare gli stores di Ca2+ intracellulari senza influenzare la risposta dei neuroni al KCl. L’applicazione di CPA non influenzava né la soglia di induzione della CSD, né la velocità di propagazione sia nei topi WT che nei topi KI. Questi risultati suggeriscono che l’intercomunicazione tra neuroni ed astrociti non sia rilevante per l’induzione e la propagazione della CSD.
La combinazione di Ca2+ imaging e patch clamp ha permesso l’osservazione, in assenza di stimolazione esterna, di attività spontanea che avviene normalmente nei networks neurali della neocorteccia; tale attività si presenta come oscillazioni lente tra periodi di depolarizzazioni indotte da attività sinaptica associate all’insorgenza di potenziali d’azione (up-states) e tra altri periodi in cui l’attività sinaptica diminuisce ed il firing scompare (down-states). Allo stesso tempo è stato possibile osservare la presenza di intense oscillazioni spontanee nella [Ca2+]int degli astrociti. Per verificare se la comunicazione reciproca tra neuroni ed astrociti potesse essere coinvolta nella generazione o nella modulazione dell’attività spontanea neuronale di network, tali oscillazioni spontanee degli astrociti sono state soppresse tramite l’applicazione di CPA. In presenza di CPA gli up-states neuronali erano ancora presenti sia nei topi WT, che nei KI, tuttavia nel primo caso si assisteva ad una riduzione significativa nella frequenza degli up-states (41 ± 3%, p = 0.0001), non riscontrabile nel caso dei topi KI (11 ± 6%, p = 0.12). Questi risultai preliminari sono in accordo con un possibile ruolo dell’intercomunicazione tra neuroni ed astrociti nell’attività spontanea dei network neuronal
Interactions between microplastics and microbiota in a One Health perspective
No full textMicroplastics are recognised as ubiquitous pollutants as they are now
found in all terrestrial and marine ecosystems. The interactions between
microbiota and microplastics are an issue of fundamental importance in
studying and maintaining global health. Microplastics alter the
structures and functions of microbial communities, resulting in adverse
health effects. A comprehensive understanding of these effects through
interdisciplinary research is essential to mitigate pollution and
protect the health of ecosystems. The review aims to explore these
interactions within a One Health framework. Indeed, a deeper
understanding of the processes involved in the interaction between
microbiota and microplastics could pave the way for new and promising
strategies to mitigate the harmful effects of microplastics on
ecosystems and human health
17beta-estradiol counteracts neuropathic pain: a behavioral, immunohistochemical, and proteomic investigation on sex-related differences in mice
Full text linkSex differences play a role in pain sensitivity, efficacy of analgesic drugs and prevalence of neuropathic pain, even if the underlying mechanisms are far from being understood. We demonstrate that male and female mice react differently to structural and functional changes induced by sciatic nerve ligature, used as model of neuropathic pain. Male mice show a gradual decrease of allodynia and a complete recovery while, in females, allodynia and gliosis are still present four months after neuropathy induction. Administration of 17 beta-estradiol is able to significantly attenuate this difference, reducing allodynia and inducing a complete recovery also in female mice. Parallel to pain attenuation, 17 beta-estradiol treated-mice show a functional improvement of the injured limb, a faster regenerative process of the peripheral nerve and a decreased neuropathy-induced gliosis. These results indicate beneficial effects of 17 beta-estradiol on neuropathic pain and neuronal regeneration and focuses on the importance of considering gonadal hormones also in clinical studies
Urinary Peptidomic Biomarkers in Kidney Diseases
Full text linkIn order to effectively develop personalized medicine for kidney diseases we urgently need to develop highly accurate biomarkers for use in the clinic, since current biomarkers of kidney damage (changes in serum creatinine and/or urine albumin excretion) apply to a later stage of disease, lack accuracy, and are not connected with molecular pathophysiology. Analysis of urine peptide content (urinary peptidomics) has emerged as one of the most attractive areas in disease biomarker discovery. Urinary peptidome analysis allows the detection of short and long-term physiological or pathological changes occurring within the kidney. Urinary peptidomics has been applied extensively for several years now in renal patients, and may greatly improve kidney disease management by supporting earlier and more accurate detection, prognostic assessment, and prediction of response to treatment. It also promises better understanding of kidney disease pathophysiology, and has been proposed as a "liquid biopsy" to discriminate various types of renal disorders. Furthermore, proteins being the major drug targets, peptidome analysis may allow one to evaluate the effects of therapies at the protein signaling pathway level. We here review the most recent findings on urinary peptidomics in the setting of the most common kidney diseases
Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency
No full textIntroduction Defects in the steroid 21-hydroxylase gene (CYP21A2) cause
21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal
hyperplasia (CAH). The disease shows a broad spectrum of clinical forms,
ranging from severe or classical (salt wasting, SW, and simple
virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects
1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC
form. There are many studies reporting the frequency of CYP21A2
pathogenic variants in different populations; however, few of them
provide comprehensive information about Italian patients. Here, we
present genetic results from a cohort of 245 unrelated Italian
individuals with clinical diagnosis of CAH due to 21OHD.
Methods A specific polymerase chain reaction (PCR) protocol combined
with Sanger sequencing was used for CYP21A2 analysis. The multiplex
ligation-dependent probe amplification (MLPA) assay was employed for
copy number variation (CNV) determination.
Results One hundred fourteen (46.5\%) of the index cases had the NC
form, 57 (23.3\%) had the SV form, and 74 (30.2\%) presented the SW form
of the disease. The most prevalent variant found in NC patients was the
p.Val282Leu (51.3\%), while the most frequent variants in the classical
form were p.Ile173Asn (8.6\%) and c.293-13C>G (26.0\%). In our study,
the frequency of large rearrangements was 15.3\%, with CAH-X alleles
representing 40\% of all DEL/CONV. In addition, 12 alleles carried rare
variants, and 1 had a novel variant p.(Arg342Gln).
We observed phenotype-genotype correlation in 94.7\% of cases. A
complete concordance was observed in Groups 0 (enzyme activity
completely impaired) where all patients had the SW form as expected. In
Group A (0-1\% residual enzyme activity), 78.4\% of patients had the
anticipated SW form while 21.6\% were diagnosed with the SV form. Within
Group B (similar to 2\% residual enzyme activity), 93.4\% of patients
exhibited SV form and 6.5\% SW disease. Finally, 92.6\% and 7.4\% of
patients belonging to Group C (enzyme partially impaired to similar to
20-60\% residual activity) exhibited NC and SV phenotypes, respectively.
Conclusion This work, representing a comprehensive genetic study,
expanded the CYP21A2 variants spectrum of Italian patients with 21OHD
and could be helpful in prenatal diagnosis and genetic counseling
Applications of MALDI-TOF mass spectrometry in clinical proteomics
Full text linkIntroduction: The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics.Areas covered: This review aims to provide an updated overview of MALDI-TOF MS applications in clinical proteomics. The most relevant features of this analysis have been discussed, highlighting both pre-analytical and analytical factors that are crucial in proteomics studies. Particular emphasis is placed on biofluids proteomics for biomarkers discovery and on recent progresses in clinical microbiology, drug monitoring, and minimal residual disease (MRD).Expert commentary: Despite some analytical limitations, the latest technological advances together with the easiness of use, the low time and low cost consuming and the high throughput are making MALDI-TOF MS instruments very attractive for the clinical practice. These features offer a significant potential for the routine of the clinical laboratory and ultimately for personalized medicine
A metaproteomic pipeline to identify newborn mouse gut phylotypes
No full textIn order to characterize newborn mouse gut microbiota phylotypes in very early-life stages, an original metaproteomic pipeline, based on LC-MS(2)-spectra and Mascot driven NCBI non-redundant repository database interrogation was developed. An original computational analysis assisted in the generation of a taxonomic gut architecture from protein hits to operational taxonomic units (OTUs) and related functional categories. Regardless of the mouse's genetic background, a prevalence of Firmicutes (Lactobacillaceae) and Proteobacteria (Enterobacteriaceae) was observed among the entire Eubacteria taxonomic node. However, a higher abundance of Firmicutes was retrieved for Balb/c gut microbiota compared to Rag2(ko) mice, the latter was mainly characterized by a Proteobacteria enriched microbiota. The metaproteomic-obtained OTUs were supported, for the identification (ID) of the cultivable bacteria fraction, corroborated by axenic culture-based MALDI-TOF MS IDs. Particularly, functional analysis of Rag2(ko) mice gut microbiota proteins revealed the presence of abundant glutathione, riboflavin metabolism and pentose phosphate pathway components, possibly related to genetic background. The metaproteomic pipeline herein presented may represent a useful tool to investigate the highly debated onset of the human gut microbiota in the first days of life, when the bacterial composition, despite its very low diversity (complexity), is still very far from an exhaustive description and other complex microbial consortia.
BIOLOGICAL SIGNIFICANCE:
The manuscript deals with a "frontier" topic regarding the study of the gut microbiota and the application of a metaproteomic pipeline to unveil the complexity of this fascinating ecosystem at the very early stages of life. Indeed during these phases, its diversity is very low but the bacterial content is highly "instable", and the relative balance between mucosal and fecal bacteria starts its dynamics of "fight" to get homeostasis. However, in the neonatal period, especially immediately after birth, a comprehensive description of this microbial eco-organ is still lacking, while it should be mandatory to highlight its first mechanisms of homeostasis and perturbation, while it co-develops with and within the host species. In order to unravel its low but almost unknown microbial community multiplicity, the newborn mouse gut, characterized by a "very" low complexity, was herein selected as model to design a LC-MS(2)-based shotgun metaproteomic approach, potentially suitable to study onset and shaping in human newborns. A microbiological semi-automatic computational analysis was performed to infer gut phylotypes; such as proof of evidence, related OTUs were compared to axenic-culture-based MALDI-TOF MS IDs showing consistency at family and phyla levels for the bacterial cultivable fraction. This article is part of a Special Issue entitled: Trends in Microbial Proteomic
p63 Isoforms Regulate Metabolism of Cancer Stem Cells
No full textp63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768
Towards a functional definition of the mitochondrial human proteome
Full text linkThe mitochondrial human proteome project (mt-HPP) was initiated by the Italian HPP group as a part of both the chromosome-centric initiative (C-HPP) and the "biology and disease driven" initiative (B/D-HPP). In recent years several reports highlighted how mitochondrial biology and disease are regulated by specific interactions with non-mitochondrial proteins. Thus, it is of great relevance to extend our present view of the mitochondrial proteome not only to those proteins that are encoded by or transported to mitochondria, but also to their interactors that take part in mitochondria functionality. Here, we propose a graphical representation of the functional mitochondrial proteome by retrieving mitochondrial proteins from the NeXtProt database and adding to the network their interactors as annotated in the IntAct database. Notably, the network may represent a reference to map all the proteins that are currently being identified in mitochondrial proteomics studies
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